Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.1.3.5 (5'-nucleotidase)
3,167 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Whole sheets of plasma membrane, each with their attached flagellum, were purified from Trypanosoma brucei. The method devised for their isolation included a new technique of cell breakage that used a combination of osmotic stress followed by mechanical sheer and avoided the problem of extreme vesiculation as well as the trapping of organelles in cell 'ghosts'. The purified membranes all contained the pellicular microtubular array. The antigenic surface coat was completely released from the plasma membrane during the isolation procedure. The membranes had a very high cholesterol/phospholipid ratio (1.54). A large proportion (42%) of the cellular DNA was recovered in the plasma-membrane fraction unless a step involving deoxyribonuclease treatment, which decreased the DNA content to less than 13%, was included before secrose-density gradient centrifugation. This step also aided the separation of plasma membranes from other cellular components. The ouabain-sensitive Na+ + K+-stimulated adenosine triphosphatase and adenylate cyclase co-purified with the plasma membranes. Although 5'-nucleotidase was thought to be a plasma-membrane component, it was easily detached from the membrane. The purified membranes were essentially free of L-alanine-alpha-oxoglutarate aminotransferase, L-asparte-alpha-oxoglutarate aminotransferase, malate dehydrogenase, oligomycin-sensitive adenosine triphosphatase, glucose 6-phosphatase, Mg2+-stimulated p-nitrophenyl phosphatase and catalase.
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PMID:The isolation and partial characterization of the plasma membrane from Trypanosoma brucei. 48 94

Evidence is presented for a direct interaction of the intrinsic membrane protein 5'-nucleotidase (5'-ribonucleotide phosphohydrolase, EC 3.1.3.5) purified from avian smooth muscle (chicken gizzard) and the cytoskeletal component actin. Two different modes of interaction can be discerned: firstly, an immediate inhibitory effect of preferentially filamentous actin (F-actin) on the enzymic (i.e., AMPase) activity of 5'-nucleotidase and a direct binding of this enzyme to immobilized F-actin. Since these effects are suppressed by the addition of myosin subfragment 1, binding of 5'-nucleotidase appears to occur along the F-actin filament axis. Secondly, a time- and 5'-nucleotidase concentration-dependent transformation of also preferentially F-actin into a form unable to inhibit the enzymic activity of deoxyribonuclease I (DNAase I). This desensitization of actin versus DNAase I is not due to a denaturation process and was found to be reversible after addition of ATP. Furthermore, it does not seem to effect the ability of actin to bind to DNAase I. The transformation is accompanied by the hydrolysis of actin-bound nucleotide into adenosine, which remains bound to actin. Therefore, the desensitization of actin versus DNAase I appears to be due to a nucleotide-dependent conformational change of actin. An unidentified contamination of the 5'-nucleotidase preparations to a varying degree with ADPase and ATPase activities appears to be responsible for the desensitization process, although a synergistic role of these activities and 5'-nucleotidase cannot be excluded.
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PMID:The interaction of 5'-nucleotidase purified from chicken gizzard and actin, and the reversible loss of the inhibitory capacity of actin on deoxyribonuclease I. 298

The transmembrane topography of the rat hepatocyte ectoenzyme 5'-nucleotidase was studied by the use of glycoprotein labelling and limited-proteolysis techniques. Comparison, by one-dimensional peptide mapping, of enzyme iodinated from outside the cell with that iodinated in the solubilized state showed that no additional iodination sites were revealed on solubilization. Incubation of newly synthesized enzyme in a microsomal membrane fraction with proteinase showed that the entire molecule of 5'-nucleotidase was protected from proteolysis. These data suggest that little, if any, of the 5'-nucleotidase molecule is present on the cytoplasmic side of the plasma membrane. No evidence was found for a previously proposed interaction between 5'-nucleotidase and actin, although the ability of preparations of 5'-nucleotidase to prevent inhibition of deoxyribonuclease I by actin was explained by minute traces of ATPase activity. Comparison of peptide maps of enzyme labelled by iodination or by methods specific for carbohydrate showed that in both cases predominantly one section of the molecule was labelled. It is proposed that the enzyme is a short-stalked integral membrane protein without a cytoplasmic domain in which about one-third of the molecule forms the accessible molecular surface.
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PMID:The membrane topography of ecto-5'-nucleotidase in rat hepatocytes. 301 17

Since evidence indicates that phorbol ester-induced production of interleukin 2 requires transcription, we investigated the possibility that the phorbol ester receptor acts directly in the nuclei of EL4 thymoma cells. Using a procedure that minimized plasma membrane contamination (as measured by 5'-nucleotidase activity) and maintained the integrity of the double nuclear membrane, we were unable to detect specific binding of [3H]phorbol 12,13-dibutyrate in nuclei of unstimulated cells. Treatment of cells with phorbol 12,13-dibutyrate (100 nM, 37 degrees C) for up to 6 h did not cause appearance of phorbol ester binding capacity in nuclei (4 +/- 8% of homogenate value; 5'-nucleotidase activity = 10 +/- 3%) despite translocation of 40% of the cytosolic binding capacity to the plasma membrane fraction. The failure to detect nuclear binding capacity in treated cells was not due to occupation of nuclear sites with unlabeled ligand; effective exchange binding was demonstrated by recovery of total homogenate binding capacity in treated cells of 82 +/- 13% of that in untreated cells. Treatment of isolated nuclei with DNase to liberate DNA binding proteins also failed to reveal any nuclear phorbol ester binding capacity. Assay of nuclei for protein kinase C enzymatic activity gave similar negative results. These data argue strongly against a direct action of the intact phorbol ester receptor (or the phorbol ester binding fragment) in the transcriptional activation of interleukin 2 in EL4 cells but cannot rule out the possibility of a role for the catalytic fragment.
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PMID:Absence of protein kinase C in nuclei of EL4 mouse thymoma cells. 303 47

We have isolated and purified, with good yields, nuclear envelopes from an androgen-responsive and from two androgen-unresponsive cell lines of the Shionogi mouse mammary carcinoma after subjecting purified nuclei to DNase at high pH and characterized them morphologically, chemically, and enzymatically. Phase-contrast microscopy revealed the nuclei to be free of cytoplasmic tags and that the nuclear envelopes were isolated as membrane "ghosts." Electron micrographs clearly showed the double-membrane system with nuclear pore complexes which illustrates that the nuclear envelopes were ultrastructurally intact. The nuclear envelopes contained little DNA, low levels of arylesterase or acid phosphatase activity, and undetectable levels of succinate dehydrogenase and 5'-nucleotidase activity. Coomassie blue staining of the nuclear envelope fractions on sodium dodecyl sulfate-polyacrylamide gels for all three cell lines revealed that most of the polypeptides were similar. However, we have identified androgen-dependent peptides of molecular weights 29 000, 32 000, and 34 000 in nuclear envelopes of the androgen-responsive cell line peptide profiles by comparing the nuclear envelopes prepared from the androgen-responsive cell line grown in intact mice, in castrated mice, and in mice which had been injected with testosterone after castration. Further investigation of the androgen regulation of these nuclear envelope peptides may help us understand the molecular mechanisms involved during morphological changes of the nucleus which occur in response to different hormonal environments.
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PMID:Isolation and characterization of nuclear envelopes from three variant cell lines of the Shionogi mouse mammary carcinoma: identification of androgen-dependent peptides. 383 Mar 47

A rapid and simple method is described to determine the orientation of isolated plasma membrane vesicle populations. The method is based on quantitation of the exposure of actin, an extrinsic membrane component bound exclusively to the cytoplasmic surface. Actin accessibility was determined using DNase I as a nonpermeating probe. The activity of this enzyme is lost upon complexation with actin, and inhibition can be conveniently monitored as the change in the rate of DNase-mediated hyperchromic reaction. Exposed actin is determined in intact vesicles, and total actin in detergent-lysed membranes; the ratio of these values equals the sum of inside-out and leaky vesicles. Sealed right-side-out vesicles are calculated by difference. Using thymocyte plasma membrane vesicles as a model system, we determined that 54% of the membrane is sealed in a right-side-out configuration. This value is consistent with independent determinations of inside-out membranes in the same preparation, obtained by 5'-nucleotidase latency measurements, and with reported values in other lymphocyte membrane preparations.
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PMID:Measurement of sidedness of isolated plasma-membrane vesicles: quantitation of actin exposure by DNase I inactivation. 622 39

5'-Nucleotidase activity of normal human embryonic lung fibroblasts (IMR-90) was found to be inhibited by the homogenates of seven different cell lines originated from patients with different kinds of leukemia and of fresh lymphocytes from a patient with Sezary syndrome (circulating T-cell lymphoma). About 97% of the inhibiting activity was found in the soluble fraction of RPMI 8402 cells, a cell line originated from the lymphocytes of a patient with acute lymphocytic leukemia. This inhibiting activity was not destroyed by dialysis, heating at 56 degrees C for 30 min, nor digestion with RNAase or DNAase. About 85% of the inhibiting activity was destroyed by digestion with papain at 37 degrees C for 1 h and it was destroyed completely by heating at 100 degrees C for 30 min. When the heated (56 degrees C for 30 min) soluble fraction of RPMI 8402 cells was mixed with the homogenate of IMR-90 cells, it had no effect on the activities of alkaline, neutral or acid phosphatases, nor of N-acetyl-beta-D-glucosaminidase or cytochrome c oxidase of IMR-90 cells. Preincubating the mixed samples for 1, 20 and 45 min, respectively, before adding the substrate, the heated soluble fraction of RPMI 8402 cells did not increase the percentage of inhibition for 5'-nucleotidase of the homogenate of IMR-90 cells. No inhibition of other enzyme activities was observed under similar conditions. These data suggest that the inhibiting activity is due to a protein(s) that is not a protease. The inhibiting activity was found in a single peak after the soluble fraction was fractionated by Sephadex G-100 chromatography and sedimentation centrifugation. The molecular weight of the inhibitor was found to be approx. 35,000 by comparing its retention volume and sedimentation rate with those of proteins of known molecular weight. The present study suggest that the previously reported undetectability of 5'-nucleotidase in permanent cell lines could be due to the presence of a protein inhibitor for 5'-nucleotidase in these human leukemic cell lines. It also supports the hypothesis that the increased 5'-nucleotidase activity in normal senescent cells in vitro may be a control in cellular aging that is missing from leukemic cells in vitro.
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PMID:Implications of a 5'-nucleotidase inhibitor in human leukemic cells for cellular aging and cancer. 630 89

In the framework of the international project aimed at the sequencing of the Bacillus subtilis genome, a 40.6 kb chromosome segment, which contains the tre locus, has been cloned and sequenced. This region (40 601 bp; 73 degrees-76 degrees on the genetic map) contains 38 complete ORFs and one partial one. Three ORFs, the closest to the hsdC locus, correspond to the treP, treA and treR genes encoding enzyme IITre, trehalose-6-phosphate hydrolase and the repressor of the tre operon, respectively. A homology search for the products deduced from the 39 ORFs revealed that 23 exhibit significant similarity to known proteins, e.g. proteins involved in acetoin utilization, deoxyribonuclease, methyladenine glycosidase, hydroxyisobutyrate dehydrogenase, multidrug resistance proteins, protein phosphatase, cyclic-nucleotide phosphodiesterase, 5'-nucleotidase and NADP(H)-flavin oxidoreductase. Based on the gene organization and the results of the homology search, it is predicted that YfjG, YfjH, YfjI, YfjJ and YfjK form an acetoin dehydrogenase system (acetoin regulatory protein, and acetoin dehydrogenase components/subunits E3, E2, E1 beta and E1 alpha respectively). yfkN, an extremely large ORF comprising 4386 nucleotides, seems to correspond to the fusion of the genes for 2',3'-cyclic-nucleotide 2'-phosphodiesterase and 5'-nucleotidase precursor.
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PMID:Cloning and sequencing of a 40.6 kb segment in the 73 degrees-76 degrees region of the Bacillus subtilis chromosome containing genes for trehalose metabolism and acetoin utilization. 896 3

Bacterial phospholipases C are known to act on biomembranes, since they can cleave the phosphodiester linkage between the polar head and the hydrophobic moiety of each phospholipid in these membranes. These enzymes have been classified into three groups; phosphatidylcholine (PC)-, sphingomyelin (SM)- and phosphatidylinositol (PI)-degrading phospholipases C. Enzymatic properties and toxicities of these phospholipases C are reviewed, in relation to author's research. Studies on the hemolytic phospholipases of Clostridium sp., Bacillus cereus etc., revealed that hydrolysis of choline-containing phospholipids such as PC and SM was responsible for the hemolysis of mammalian erythrocytes by these enzymes in the presence of Ca2+ and/or Mg2+. Also, the studies on a structure-activity correlation of SM-hydrolyzing phospholipase C from B. cereus disclosed the similarity of active sites between this enzyme and bovine pancreatic DNase I. By action of PI-degrading phospholipases C, several membrane proteins such as alkaline phosphatase, 5'-nucleotidase, VSG (protozoal surface glycoprotein) etc., were shown to be released from the plasma membranes of eucaryotic cells. From structural analysis, these proteins have been revealed to be glycosylphosphatidylinositol (GPI)-anchored proteins bound to the plasma membranes with carboxyl terminal-attached glycolipid. Biochemistry and molecular biology of GPI-anchored proteins, including the structures and biosynthetic routes of GPI glycolipids as well as the process of GPI attachment to proteins, requirements of C-terminal signal peptide for the protein modification by GPI, and distribution of GPI-anchored proteins in living world, are described in relation to our studies.
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PMID:[Development from actions of bacterial phospholipases C on eucaryotic plasma membranes to molecular biology of GPI-anchored proteins]. 1043

Among the myriad of enzymes present in animal venoms, nucleotidases and nucleases are poorly investigated. Herein, we studied such enzymes in 28 crude venoms of animals found in Brazil. Higher levels of ATPase, 5'-nucleotidase, ADPase, phosphodiesterase and DNase activities were observed in snake venoms belonging to Bothrops, Crotalus and Lachesis genera than to Micrurus genus. The venom of Bothrops brazili snake showed the highest nucleotidase and DNase activities, whereas that of Micrurus frontalis snake the highest alkaline phosphatase activity. On the other hand, the venoms of the snake Philodryas olfersii and the spider Loxosceles gaucho were devoid of most nucleotidase and DNase activities. Species that exhibited similar nucleotidase activities by colorimetric assays showed different banding pattern by zymography, suggesting the occurrence of structural differences among them. Hydrolysis of nucleotides showed that 1 mol of ATP is cleaved in 1 mol of pyrophosphate and 1 mol of orthophosphate, whereas 1 mol of ADP is cleaved exclusively in 2 mol of orthophosphates. Pyrophosphate is barely hydrolyzed by snake venoms. Phosphodiesterase activity was better correlated with 5'-nucleotidase, ADPase and ATPase activities than with DNase activity, evidencing that phosphodiesterases are not the main agent of DNA hydrolysis in animal venoms. The omnipresence of nucleotidase and DNase activities in viperid venoms implies a role for them within the repertoire of enzymes involved in immobilization and death of preys.
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PMID:Nucleotidase and DNase activities in Brazilian snake venoms. 1790 25


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